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Abstract: TH-OR071

The Role of Megalin in the Regulation of Renal RAS: Investigation Using LC/MS-Based Multiple-Reaction Monitoring Quantification of Angiotensin Peptides

Session Information

Category: Hypertension and CVD

  • 1403 Hypertension and CVD: Mechanisms

Authors

  • Goto, Sawako, Niigata University , Chuo-ku, niigata, Japan
  • Yoshida, Yutaka, Niigata University , Chuo-ku, niigata, Japan
  • Hosojima, Michihiro, Niigata University , Chuo-ku, niigata, Japan
  • Iida, Tomomichi, Niigata University , Chuo-ku, niigata, Japan
  • Kuwahara, Shoji, Niigata University , Chuo-ku, niigata, Japan
  • Kabasawa, Hideyuki, Niigata University , Chuo-ku, niigata, Japan
  • Kaseda, Ryohei, Niigata University , Chuo-ku, niigata, Japan
  • Narita, Ichiei, Niigata University , Chuo-ku, niigata, Japan
  • Saito, Akihiko, Niigata University , Chuo-ku, niigata, Japan
Background

The mechanisms underlying the regulation of the renal renin-angiotensin system (RAS) are largely unknown. Megalin, an endocytosis receptor in proximal tubular cells, reabsorbs angiotensinogen (AGT) and angiotensin (Ang) peptides filtered through glomeruli. We have developed liquid chromatography (LC)/mass spectrometry (MS)-based multiple-reaction monitoring quantification of Ang peptides. This study aimed to investigate the role of megalin in the renal RAS by measuring Ang peptides in biological samples obtained from kidney-specific, tamoxifen-inducible conditional megalin knock-out mice (KO) and their controls (Ctl).

Methods

Stable isotope-labeled Ang peptides were added to each sample as internal standards. Under the steady condition and 30 min after AGT administration, Ang peptides were extracted by reverse phase chromatography and evaluated by LC/MS-based quantification.

Results

Under the steady condition, Ang-I and -II were quantified in the kidney and plasma without significant differences between KO and Ctl (e.g., kidney Ang-II: 573±257 in KO and 569±172 fmol/g in Ctl). High levels of Ang-I, -II, -1-7 and -1-9 were detected in KO’s fresh urine (e.g., Ang-II: 3.8±4.5 pmol/mL), whereas their concentrations were below the limit of detection in Ctl. AGT administration increased kidney Ang peptides without significant increase in plasma. Kidney Ang-II and -1-9 levels in KO administered with AGT were significantly lower than those in Ctl (e.g., Ang -II: 1760±1043 in KO and 2489±836 fmol/g in Ctl, p<0.05); kidney Ang-I showed a similar tendency, whereas there was no difference in Ang -1-7.

Conclusion

High-level excretion of Ang peptides in KO’s urine indicated that they were produced in the tubular lumen. AGT administration increased kidney Ang peptides independently of the systemic RAS. The increase of kidney Ang-I, -II, and -1-9 induced by AGT administration was suppressed in KO compared with Ctl, whereas there was no difference in Ang-1-7. Ang-I, -II and -1-9 generated in the tubular lumen may be excreted more in KO than Ctl or converted to Ang-1-7. Megalin blockade could be beneficial for modulating the renal RAS by regulating the renal handling of AGT and Ang peptides.